Regulatory proteins have a central role in health and disease: DNA-binding proteins control development, differentiation and cell growth. The "helix-turn-helix" unit is one of the most important structural motifs involved in protein-DNA interactions. It was discovered in a set of prokaryotic repressors, but the eukaryotic homeodomain proteins have been characterized, and they play a central role in development, specification of cell type, and the largest and most important families of eukaryotic regulatory proteins. Our first goal is to understand how this superfamily of "helix-turn-helix" proteins recognizes their binding sites and how the bound proteins regulate gene expression. We will continue with our structural analysis of the lambda repressor-operator interactions, studying complexes with altered repressors and altered binding sites. We will crystallize and cocrystallize representative homeodomains-from yeast, from Drosophila, and from humans-and will use x-ray crystallography to determine the structures of these proteins and complexes. As these projects are completed, we will begin structural studies of other important motifs involved in eukaryotic gene regulation. We are particularly interested in the "helix-loop-helix" proteins that are involved in differentiation and/or sex determination. We plan to crystallize MyoD (a helix-loop-helix protein that is involved in the differentiation of muscle cells) as a complex with the appropriate DNA sites. Analysis and comparison of these major families of DNA-binding proteins should give us a much deeper understanding of the molecular basis for site- specific recognition and gene regulation.